乙型肝炎病毒P区RNA干扰抑制2.2.15细胞乙型肝炎病毒表面抗原和e抗原分泌的实验研究

Inhibition of HBsAg and HBeAg secretion by RNA interference of the polymerase gene sequence of hepatitis B virus: an experimental study

目的观察针对乙型肝炎病毒(HBV)P区基因的RNA干扰(RNAi)表达载体pGE-HBVP在HepG2.2.15(2.2.15)细胞中抑制HBV表面抗原(HBsAg)和e抗原(HBeAg)的分泌效应,以阐明HBV P区小干扰RNA(siRNA)抑制HBV复制和蛋白表达的影响。方法构建并鉴定pGE-HBVP,将此载体转染至完整HBV复制模型2.2.15细胞,用化学发光免疫检测法检测并分析转染后不同时间段细胞培养上清中HBsAg和HBeAg的含量变化,并用免疫细胞化学方法观察转染后细胞中HBsAg的表达变化。结果成功构建了5个针对HBV P区的RNAi表达载体pGE-HBVP1～pGE-HBVP5,其中pGE-HBVP1和pGE-HBVP2具有明显的HBsAg和HBeAg分泌抑制效应和表达抑制效应。抑制率最高的pGE-HBVP2在转染2.2.15细胞效率为30%～40%的基础上,转染后24、48、72和96h培养上清中的HBsAg分泌抑制率分别为28.88%、32.28%、29.10%和18.42%,HBeAg的分泌抑制率分别为38.33%、27.50%、33.41%和12.60%。细胞免疫细胞化学结果显示,pGE-1空载体转染后2.2.15细胞的HBsAg的表达阳性率约为82%,而pGE-HBVP2转染后2.2.15细胞的HBsAg的表达阳性率约为50%,和pGE-1空载体相比阳性率显著下降。结论针对HBV P区的RNAi可以抑制HBV病毒抗原的分泌和表达。

Objective To investigate the effects of small interfering RNA （siRNA） targeting the polymerase （P） gene sequence of hepatitis B virus （HBV） on the replication and antigen secretion of HBV. Methods From the 29 base sequences of the HBV in the HepG2.2.15 cells that accord with the demands of siRNA designing five sequences targeting the P gene of HBV were selected and cloned into the siRNA expressing vector pGE-1. Then the plasmid pGE-HBVP was transfected into the cultured HepG2.2.15 ceils. Chemiluminescent immunoassay was used to determine the levels of HBsAg and HBeAg in the supernatant of culture medium 24, 48, 72, and 96 hours after the transfection and the expression of HBsAg in the 2.2.15 cells 24 hours after the transfeetion so as to observe the inhibitory effects. Untransfeeted cells and cells transfected with blank pGE-1 vector were used as controls. Results Five vectors expressing the siRNAs targeting the HBV P region, pGE-HBVPI-pGE-HBV5 were successfully constructed. The efficiency of transfection of the vectors into the 2.2. 15 cells were 30% to 40%. 24, 48, 72, and 96 hours after the transfection of pGE-HBVP2, the strongest inhibitor among the five, the inhibitory rates of HBsAg secretion in the supernatant were 28.88% , 32.28% , 29.10% , and 18.42% respectively; and the inhibitory rates of HBeAg secretion were 38.33%, 27.50% , 33.41% , and 12.60% respectively. In view of the transfeetion efficiency of 30% - 40% , the actual inhibitory rate of HBV antigen secretion might reach 80% and over. 24 hours after the transfection the expression rate of HBsAg in the 2.2. 15 cells transfected with pGE-HBVP2 was 50%, significantly lower than that in the cells transfected with the blank vector pGE-1 （82%）. Conclusion siRNAs targeting the HBV P gene effectively prevent the HBV gene expression and replication and may play an important role in the clinical anti-viral treatment.